The products of ras genes comprise a family of guanine nucleotide binding proteins that are involved in the control of eukaryotic cell proliferation. Specific point mutations result in ras oncoproteins which have the ability to neoplasticly transform mammalian cells, and activated ras genes have been observed in at least 10% of all human tumors. Their incidence in certain malignancies, such as in colorectal and pancreatic cancers, is far greater.
Genetic studies first established that ras proteins, referred to as ras p21, must be formed by post-translational modification of a precursor protein with a defined carboxy-terminal structure, in order to exert their biological function. This structure, known as the CAAX box, is formed of a conserved cysteine residue located four amino acid-residues from the carboxy terminus, which in the case of ras is position 186 (except in the K-ras4B p21 protein, in which cysteine is located at position 185), followed by two aliphatic amino acids and any carboxy-terminal amino acid residue. Mutations affecting the basic CAAX box structure of oncogenic ras p21 proteins completely abolish their transforming activity, presumably by impeding their interaction with the inner side of the plasma membrane. Such interaction requires a series of post-translational modifications within the CAAX box motif which include (a) farnesylation of the cys residue of the CAAX box; (b) cleavage of the three carboxy-terminal amino acid residues; and (c) methylation of the free carboxyl group generated in the resulting carboxy-terminal farnesyl-cysteine residue. The interaction of these farnesylated ras p21 proteins with cellular membranes in some cases is further strengthened by palmitoylation of neighboring upstream cysteine residues. See Hancock, et al, Jun. 30, 1989, Cell 57:1167-1177; and Casey, et al, November 1989, Proc. Natl. Acad. Sci. U.S.A. 86:8323-8327.
Recent studies have suggested that the donor of the farnesyl residue present in ras p21 proteins is farnesyl pyrophosphate (FPP), a precursor also in the biosynthesis of cholesterol. The transfer of the farnesyl group from FPP, the donor molecule, to ras proteins is mediated by the enzyme, protein-farnesyl transferase (FT).
Treatment of S. cerevisiae cells or Xenopus oocytes with inhibitors of HMG-CoA reductase, the enzyme responsible for the synthesis of mevalonic acid, the precursor of isoprenoid compounds, blocks the function of ras proteins in these cells. These results have raised the possibility of using inhibitors of cholesterol biosynthesis, that is, HMG CoA reductase inhibitors, to block neoplastic transformation induced by ras oncogenes. See, Schafer, et al, Jul. 28, 1989, Science 245:379-385; and Goldstein and Brown, Feb. 1, 1990, Nature 343:425-430.
Rine and Kim, "A Role for Isoprenoid Lipids in the Localization and Function of an Oncoprotein," The New Biologist, Vol. 2, No. 3 (March), 1990: pp 219-236, disclose at pages 222-223 that "lovastatin [also known as Mevacor], compactin, and related drugs that have been developed for the treatment of hypercholesterolemia act by inhibiting 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase), the enzyme that catalyzes the rate-limiting step in the synthesis of cholesterol and all other polyisoprenoids . . . . The drugs were tested in the Xenopus oocyte assay . . . for their ability to pharmacologically suppress activated H-Ras.sup.va112 . . . . These experiments pinpointed farnesyl pyrophosphate as the likely donor molecule for farnesylation of Ras protein, and suggested a rationale for a novel pharmacological route to block the action of this important human oncoprotein."
"Earlier work had already provided evidence that inhibition of isoprenoid synthesis by use of inhibitors of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase could slow the growth of tumors in animals. In particular, continuous, high levels of lovastatin caused substantial growth inhibition of a mouse neuroblastoma . . . . Although the oncogene(s) responsible for this tumor has not yet been identified and the dosage required to suppress the tumor was rather high, this study does support the notion that protein prenyl transferase(s) responsible for Ras modification might serve as useful targets for chemotherapy . . . ."
U.S. patent application Ser. No. 520,570 filed May 8, 1990, by Barbacid et al discloses protein-farnesyl transferase (FT) assays for identifying compounds that block the farnesylation of ras oncogene products. The Barbacid et al invention is based, in part, on the discovery and identification of the FT enzyme which catalyzes the transfer of the farnesyl group from the donor, farnesyl pyrophosphate (FPP), to the ras p21 Cys.sup.186 residue. Farnesylation of ras proteins is required for their attachment to the inner cell membrane and biological activity. Farnesylation of ras oncogene products is required for ras mediated transforming activity. Because the assays of the Barbacid et al invention are designed to target a step subsequent to the synthesis of FPP (in the cholesterol chain), they allow for the identification of compounds that interfere with farnesylation of the ras oncogene products and inhibit their transforming activity, yet do not interfere with the synthesis of FPP, a precursor in the synthesis of cholesterol, ubiquinones, dolichols and Haem A. Therefore, FT inhibitory compounds that do not disrupt important cellular pathways which require FPP may be identified using the Barbacid et al assay.
Squalene synthetase is a microsomal enzyme which catalyzes the reductive dimerization of two molecules of farnesyl pyrophosphate (FPP) in the presence of nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH) to form squalene (Poulter, C. D.; Rilling, H. C., in "Biosynthesis of Isoprenoid Compounds," Vol. I, Chapter 8, pp. 413-441, J. Wiley and Sons, 1981, and references therein). This enzyme is the first committed step of the de novo cholesterol biosynthetic pathway.
Squalene synthetase inhibitors which block the action of squalene synthetase (after the formation of farnesyl pyrophosphate) are disclosed in U.S. Pat. Nos. 4,871,721 and 5,025,003, U.S. application Ser. No. 501,204, filed Mar. 29, 1990, and U.S. application Ser. No. 699,429, filed May 13, 1991.